The present invention is in the field of molecular biology, enzymology, biochemistry and clinical medicine. In particular, the present invention provides a human recombinant xcex1-L-iduronidase, methods of large-scale production and purification of commercial grade human recombinant xcex1-L-iduronidase enzyme, and methods to treat certain genetic disorders including xcex1-L-iduronidase deficiency and mucopolysaccharidosis I (MPS I).
Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates are structural components of the human body covalently attached to numerous other entities such as proteins and lipids (called glycoconjugates). For example, human connective tissues and cell membranes comprise proteins, carbohydrates and a proteoglycan matrix. The carbohydrate portion of this proteoglycan matrix provides important properties to the body""s structure.
A genetic deficiency of the carbohydrate-cleaving, lysosomal enzyme xcex1-L-iduronidase causes a lysosomal storage disorder known as mucopolysaccharidosis I (MPS I) (Neufeld and Muenzer, pp. 1565-1587, in The Metabolic Basis of Inherited Disease, Eds., C. R. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, McGraw-Hill, New York (1989)) In a severe form, MPS I is commonly known as Hurler syndrome and is associated with multiple problems such as mental retardation, clouding of the cornea, coarsened facial features, cardiac disease, respiratory disease, liver and spleen enlargement, hernias, and joint stiffness. Patients suffering from Hurler syndrome usually die before age 10. In an intermediate form known as Hurler-Scheie syndrome, mental function is generally not severely affected, but physical problems may lead to death by the teens or twenties. Scheie syndrome is the mildest form of MPS I. It is compatible with a normal life span, but joint stiffness, corneal clouding and heart valve disease cause significant problems.
The frequency of MPS I is estimated to be 1:100,000 according to a British Columbia survey of all newborns (Lowry, et al., Human Genetics 85:389-390 (1990)) and 1:70,000 according to an Irish study (Nelson, Human Genetics 101:355-358 (1990)). There appears to be no ethnic predilection for this disease. It is likely that worldwide the disease is underdiagnosed either because the patient dies of a complication before the diagnosis is made or because the milder forms of the syndrome may be mistaken for arthritis or missed entirely. Effective newborn screening for MPS I would likely find some previously undetected patients.
Except for a few patients which qualify for bone marrow transplantation, there are no significant therapies available for all MPS I patients. Hobbs, et al. (Lancet 2: 709-712 (1981)) first reported that bone marrow transplantation successfully treated a Hurler patient. Since that time, clinical studies at several transplant centers have shown improvement in physical disease and slowing or stabilizing of developmental decline if performed early. (Whitley, et al., Am. J. Med. Genet. 46: 209-218 (1993); Vellodi, et al., Arch. Dis. Child. 76: 92-99 (1997); Peters, et al., Blood 91: 2601-2608 (1998); Guffon, et al., J. Pediatrics 133: 119-125 (1998)) However, the significant morbidity and mortality, and the need for matched donor marrow, limits the utility of bone marrow transplants. An alternative therapy available to all affected patients would provide an important breakthrough in treating and managing this disease.
Enzyme replacement therapy has been considered a potential therapy for MPS I following the discovery that xcex1-L-iduronidase can correct the enzymatic defect in Hurler cells in culture, but the development of human therapy has been technically unfeasible until now. In the corrective process, the enzyme containing a mannose-6-phosphate residue is taken up into cells through receptor-mediated endocytosis and transported to the lysosomes where it clears the stored substrates, heparan sulfate and dermatan sulfate. Application of this therapy to humans has previously not been possible due to inadequate sources of xcex1-L-iduronidase in tissues.
For xcex1-L-iduronidase enzyme therapy in MPS I, a recombinant source of enzyme has been needed in order to obtain therapeutically sufficient supplies of the enzyme. The cDNA for the canine enzyme was cloned in 1991 (Stoltzfus, et al., J. Biol. Chem. 267:6570-6575 (1992) and for the human enzyme in the same year. (Scott, et al., Proc. Natl. Acad. Sci. U.S.A. 88:9695-9699 (1991), Moskowitz, et al., FASEB J 6:A77 (1992)). Following the cloning of cDNA for xcex1-L-iduronidase, the production of adequate quantities of recombinant xcex1-L-iduronidase allowed the study of enzyme replacement therapy in canine MPS I. (Kakkis, et al., Protein Expr. Purif. 5: 225-232 (1994)) Enzyme replacement studies in the canine MPS I model demonstrated that intravenously-administered recombinant xcex1-L-iduronidase distributed widely and reduced lysosomal storage from many tissues. (Shull, et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12937-12941 (1994); Kakkis, et al., Biochem. Mol. Med. 58: 156-167 (1996)).
In one aspect, the present invention features a method to mass produce human recombinant xcex1-L-iduronidase in large scale amounts with appropriate purity to enable large scale production for long term patient use of the enzyme therapy. In a broad embodiment, the method comprises the step of transfecting a cDNA encoding for all or part of an xcex1-L-iduronidase into a cell suitable for the expression thereof. In some embodiments, a cDNA encoding for a complete xcex1-L-iduronidase is used, preferably a human xcex1-L-iduronidase. However, in other embodiments, a cDNA encoding for a biologically active fragment or mutant thereof may be used. Specifically, one or more amino acid substitutions may be made while preserving or enhancing the biological activity of the enzyme. In other preferred embodiments, an expression vector is used to transfer the cDNA into a suitable cell or cell line for expression thereof. In one particularly preferred embodiment, the cDNA is transfected into a Chinese hamster ovary cell to create cell line 2.131. In yet other preferred embodiments, the production procedure features one or more of the following characteristics which have demonstrated particularly high production levels: (a) the pH of the cell growth culture may be lowered to about 6.5 to 7.0, preferably to about 6.8-7.0 during the production process, (b) as many as 2 to 3.5 culture volumes of the medium may be changed during each 24-hour period by continuous perfusion, (c) oxygen saturation may be optimized to about 40% but may be as high as 80%, (d) macroporous cellulose microcarriers with about 5% serum in the medium initially, may be used to produce cell mass followed by a rapid washout shift to protein-free medium for production, (e) a protein-free or low protein-medium such as a JRH Biosciences PF-CHO product may be optimized to include supplemental amounts of one or more ingredients selected from the group consisting of: glutamate, aspartate, glycine, ribonucleosides, and deoxyribonucleosides; (f) a stirred tank suspension culture may be perfused in a continuous process to produce iduronidase.
In a second aspect, the present invention provides a transfected cell line which features the ability to produce xcex1-L-iduronidase in amounts which enable using the enzyme therapeutically. In preferred embodiments, the present invention features a recombinant Chinese hamster ovary cell line such as the 2.131 cell line that stably and reliably produces amounts of xcex1-L-iduronidase which enable using the enzyme therapeutically. In some preferred embodiments, the cell line may contain more than 1 copy of an expression construct. In even more preferred embodiments, the cell line expresses recombinant xcex1-L-iduronidase in amounts of at least 20 micrograms per 107 cells per day.
In a third aspect, the present invention provides novel vectors suitable to produce xcex1-L-iduronidase in amounts which enable using the enzyme therapeutically. In preferred embodiments, the present invention features an expression vector comprising a cytomegalovirus promoter/enhancer element, a 5xe2x80x2 intron consisting of a murine Cxcex1 intron, a cDNA encoding all or a fragment or mutant of an a xcex1-L-iduronidase, and a 3xe2x80x2 bovine growth hormone polyadenylation site. Also, preferably the cDNA encoding all or a fragment or mutant of an xcex1-L-iduronidase is about 2.2 kb in length. This expression vector may be transfected at, for example, a 50 to 1 ratio with any appropriate common selection vector such as pSV2NEO, to enhance multiple copy insertions. Alternatively, gene amplification may be used to induce multiple copy insertions.
In a fourth aspect, the present invention provides novel xcex1-L-iduronidase produced in accordance with the methods of the present invention and thereby present in amounts which enable using the enzyme therapeutically. The specific activity of the xcex1-L-iduronidase according to the present invention is in excess of 200,000 units per milligram protein. Preferably, it is in excess of about 240,000 units per milligram protein. The molecular weight of the xcex1-L-iduronidase of the present invention is about 82,000 daltons, about 70,000 daltons being amino acid, and about 12,000 daltons being carbohydrates.
In a fifth aspect, the present invention features a novel method to purify xcex1-L-iduronidase. According to a first embodiment, a cell mass may be grown in about 5% serum-containing medium, followed by a switch to a modified protein-free production medium without any significant adaptation to produce a high specific activity starting material for purification. In one preferred embodiment, a three step column chromatography may be used to purify the enzyme. Such a three step column chromatography may include using a blue sepharose FF, a Cu++ chelating sepharose chromatography and a phenyl sepharose HP chromatography. In another preferred embodiment, an acid pH treatment step is used to inactivate potential viruses without harming the enzyme. Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl 200 columns are removed and Blue-Sepharose and copper chelating columns added to increase the capacity of the large scale purification process, to reduce undesirable leachables inappropriate for long term patient use, and to improve the purity of the product.
In a sixth aspect, the present invention features novel methods of treating diseases caused all or in part by a deficiency in xcex1-L-iduronidase. In one embodiment, this method features administering a recombinant xcex1-L-iduronidase or a biologically active fragment or mutant thereof alone or in combination with a pharmaceutically suitable carrier. In other embodiments, this method features transferring a nucleic acid encoding all or a part of an xcex1-L-iduronidase into one or more host cells in vivo. Preferred embodiments include optimizing the dosage to the needs of the organism to be treated, preferably mammals or humans, to effectively ameliorate the disease symptoms. In preferred embodiments, the disease is Mucopolysaccharidosis I (MPS I), Hurler syndrome, Hurler-Scheie syndrome or Scheie syndrome.
In a seventh aspect, the present invention features novel pharmaceutical compositions comprising xcex1-L-iduronidase useful for treating a disease caused all or in part by a deficiency in xcex1-L-iduronidase. Such compositions may be suitable for administration in a number of ways such as parenteral, topical, intranasal, inhalation or oral administration. Within the scope of this aspect are embodiments featuring nucleic acid sequences encoding all or a part of an xcex1-L-iduronidase which may be administered in vivo into cells affected with an xcex1-L-iduronidase deficiency.